Cheap and readily available industrial enzymes are the key drivers behind the industrial biotechnology revolution where chemicals and energy fuels are increasingly being produced biologically using renewable resources. In contrast to high margin enzymes or other proteins used for therapeutic and diagnostic purposes, industrial enzymes typically have lower margins and are manufactured in large quantities with limited downstream processing after fermentation. For the industrial enzyme market, the production costs are of critical importance to commercial success. In order to fulfill the increasing demand for cheaper and readily available enzymes for the renewable chemicals and energy industries, there is an urgent need for more efficient and cost-effective production processes.
Major industrial enzyme manufacturers all have significant production capacities devoted to fungal fermentation, in particular using filamentous fungi such as Aspergillus spp, Penicillium spp, Trichoderma spp., Chrysosporium lucknowense (C1) and Myceliophthora thermophila (reclassified)), to make a range of enzyme products used in the textile, detergent, food and feed, and the nascent biofuels industries (Sharma et al., World J. of Microbiol. and Biotech., 25(12):2083-2094 (2009); Visser et al., Industrial Biotech., 7(3):214-223 (2011)).
Commercial production of a protein of interest in Aspergillus and Trichoderma typically requires an inducer compound that activates the transcriptional switch under a strong native promoter driving a gene of interest expressing a targeted enzyme or protein. Insoluble substrates such as starch and cellulose are typically used in laboratory scale to induce protein expression in Aspergillus and Trichoderma, respectively. Their implementation at industrial scale, however, is hampered by poor substrate consistency and materials and handling issues encountered at large scale such as sterilization, feeding, mixing, and viscosity issues. Consequently, highly efficient soluble inducers are more preferred in the commercial production of enzymes and other proteins.
Maltose, isomaltose, and maltodextrins are commonly used as soluble inducers for the induction of alpha-amylase and glucoamylase enzymes in Aspergillus spp. (Barton et al., J. Bacteriol., b(3):771-777 (1972)), while sophorose, cellobiose, and lactose are three effective soluble inducers widely used in the industry for enzyme production in Trichoderma spp. (Kubicek et al., Biotech. Biofuels, 2:19 (2009)). Sophorose, a beta 1, 2-disaccharide, is considered to be the most powerful inducer of the cellulase gene promoter in Trichoderma reesei, being 2500 times as active as cellobiose (Mandels et al., J. Bacteriol., 83(2):400-408 (1962)). These respective soluble inducers are all metabolized by Aspergillus or Trichoderma before, during, or after induction. Therefore, they are not considered as gratuitous inducers, thus requiring them to be continually supplied during the fermentation in order to achieve optimum induction.
In industrial enzyme production through submerged fermentation, soluble inducers are typically supplied to the fermenter in a fed batch either alone or supplemented with alternative carbon sources such as glucose in a carbon-limited fashion. Due to the inducers being non-gratuitous, the induction process, if not fully optimized, will typically cause catabolic repression that significantly lowers the productivity, along with unnecessary growth that significantly lowers the yield. Thus, there exists a need in the art for a stronger, gratuitous, or nearly gratuitous inducer that decouples induction from unintended repression, growth, or other physiological functions, allowing more rigorous process optimization to significantly improve productivity and yield than is currently possible. A more powerful inducer can also be easily used to supply the induction needs of cheaper and non-inductive feedstocks as well as to enhance the induction of conventional inducers such as lactose, cellulose, maltodextrins, or cheaper alternatives such as starch and cellulose hydrolysates, and also in alternative production processes such as solid-phase fermentations.
Because carbohydrate-based inducers are both inducers and repressors for protein expression, methods to enhance the induction power by modifying the inducer to slow its uptake or metabolization are known. Hydrolysis products of cellobiose octaacetate, although not characterized, are known to carry superior induction power than cellobiose (Mandels and Reese, J. Bacteriol., 79(6):816-826 (1960)). Sucrose monopalmitate induced a sucrase yield that is 80 times that of sucrose in Pullularia pullulans (Reese et al., J. Bacteriol., 100(3):1151-1154 (1969)). Similarly, acetyl cellobioses were more effective than glucose or cellobiose at inducing cellulase in Penicillium purpurogenum, with mono-O-acetyl cellobiose being the most active inducer tested (Suto et al., J. Ferment. Bioengin., 72(5):352-357 (1991)). A recent filing by the inventor (International Application No.: WO 2013/003291) disclosed a novel class of sophorose esters derived from dilute acid treatment of natural sophorolipid that is at least a 30 times more powerful inducer than sophorose itself.